Epidermal Growth Factor Receptor (EGFR) plays an important role in cellular proliferation as well as apoptosis, angiogenesis and metastatic spread, processes that are crucial to tumour progression (Salomon et al, Crit. Rev. Oncology/Haematology, 19:183-232 (1995); Wu et al, J. Clin. Invest., 95:1897-1905 (1995); Karnes et al, Gastroenterology, 114:930-939 (1998); Woodburn et al, Pharmacol. Therap. 82: 241-250 (1999); Price et al, Eur. J. Cancer, 32A:1977-1982 (1996)). Indeed, studies have shown that EGFR-mediated cell growth is increased in a variety of solid tumours including non-small cell lung cancer, prostate cancer, breast cancer, gastric cancer, and tumours of the head and neck (Salomon D S et al, Critical Reviews in Oncology/Haematology, 19:183-232 (1995)). Furthermore, excessive activation of EGFR on the cancer cell surface is now known to be associated with advanced disease, the development of a metastatic phenotype and a poor prognosis in cancer patients (Salomon D S et al., Critical Reviews in Oncology/Haematology 19:183-232 (1995)).
Furthermore, EGFR expression is frequently accompanied by the production of EGFR-ligands, TGF-alpha and EGF among others, by EGFR-expressing tumour cells which suggests that an autocrine loop participates in the progression of these cells (Baselga, et al. (1994) Pharmac. Therapeut. 64: 127-154; Modjtahedi, et al. (1994) Int. J. Oncology. 4: 277-296). Blocking the interaction between such EGFR ligands and EGFR therefore can inhibit tumor growth and survival (Baselga, et al. (1994) Pharmac. Therapeut. 64: 127-154).
The EGFR is a membrane bound glycoprotein with a molecular weight of approximately 170 kDa. EGFR consists of a glycosylated external ligand-binding domain (621 residues) and a cytoplasmic domain (542 residues) connected by a short 23 amino acid transmembrane linker. The extracellular part of EGFR contains 25 disulfide bonds and 12 N-linked glycosylation sites, and is generally considered to consist of four sub-domains. X-ray crystal structures of the EGFR suggest that the receptor adopts both an autoinhibited tethered-conformation that cannot bind EGF (Ferguson et al, Mol Cell, 2003, vol 11: 507-517) and an active conformation that may mediate EGF ligand binding and receptor dimerisation (Garret et al, Cell 2002, vol 110:763-773; Ogiso et al, Cell, 2002, vol 110:775-787). In particular, domain I and domain III have been suggested to provide additive contributions for formation of a high-affinity ligand binding site. Domains II and IV are cysteine-rich laminin-like regions that stabilise protein folding and contain a possible EGFR dimerisation interface.
EGFR is known to exist in a number of different conformations on the cell surface, where the tethered or locked confirmation is the most frequent. The tethered conformation cannot dimerise and hence is inactive. The therapeutic antibody Erbitux is known to stabilise the tethered conformation by binding to domain III and sterically hampering the receptor in reaching the untethered state. However, some receptors may still be able to adopt the untethered conformation, bind ligand and dimerise. A monoclonal antibody (mAb) will typically only be effective in binding against one of the conformations and therefore cannot effectively target cancer cells exhibiting other conformations or cancer cells exhibiting a variety of conformations.
Monoclonal antibodies (mAbs) directed to the ligand-binding domain of EGFR can block the interaction with EGFR ligands and, concomitantly, the resultant intracellular signaling pathway.
Erbitux™ (Cetuximab) is a recombinant, human/mouse chimeric monoclonal antibody that binds specifically to the extracellular domain of the human (EGFR). Erbitux is composed of the Fv regions of a murine anti-EGFR antibody with human IgG1 heavy and kappa light chain constant regions and has an approximate molecular weight of 152 kDa. Erbitux is produced in mammalian cell culture (murine myeloma). Erbitux is approved for the treatment of patients with metastatic colorectal cancer and whose tumor expresses EGFR. In addition, Erbitux is used in combination with radiation therapy to treat patients with squamous cell cancer of the head and neck that cannot be removed by surgery or as second line treatment of squamous cell cancer of the head and neck that have failed standard platinum-based therapy.
Vectibix™ (panitumumab) is a recombinant, human IgG2 kappa monoclonal antibody that binds specifically to the human EGFR. Vectibix has an approximate molecular weight of 147 kDa. Panitumumab is produced in genetically engineered mammalian cells (Chinese Hamster Ovary). Vectibix is approved for the treatment of patients with metastatic colorectal cancer and whose tumor expresses EGFR with disease progression on or following fluoropyrimidine-, oxaliplatin-, and irinotecan-containing chemotherapy regimens.
A number of mutant EGF receptors have been identified on human tumour cells. These may render the receptor activity independently of ligand binding (EGFRvIII) leading to enhanced tumorigenicity. Monoclonal antibodies against a mutant EGFR may be generated, but such a monoclonal antibody will not necessarily be effective against non-mutated EGFR.
Mutations of EGFR have been identified in human cancer patients that affect their response to chemotherapy directed toward EGFR. WO 2006/110478 (Novartis) disclosed 43 mutations as well as 18 SNPs in the EGFR open reading frame. Some missense mutations are identified in two or more types of tumour types. WO 2006/091899 (Amgen) disclosed eight further mutations identified in various cancer cells. One or more of these mutations may be located in the epitope or affect the structure of the epitope bound by one of the currently approved monoclonal antibodies. Patients carrying such mutation(s) will not be treatable by a monoclonal antibody.
Furthermore, there are reports in literature showing heterogeneity in glycosylation of at least one of the glycosylation sites (Whitson et al., 2005 Biochemistry 44:14920-31; Zhen et al. 2003 Biochemistry 42; 5478-92). Such heterogeneity may directly or indirectly result in differential exposure of epitopes that vary among tumour cells.
Antibody dependent cellular cytotoxicity (ADCC) is an alternative mechanism by which antibodies mediate killing of tumour cells. The level of ADCC is dependent on several factors including IgG subtype (IgM>IgG1>IgG2), antibody density on target cells, antibody glycosylation pattern as well as the properties of the target itself.
Friedmann et al (PNAS 2005, 102:1915-20) have shown that two murine monoclonal antibodies selected for their ability to inhibit EGF binding to EGFR by binding distinct EGFR epitopes are able to synergistically down-regulate receptor expression in KB cells and CHO cells transiently expressing EGFR. Cross competitive EGF inhibiting antibodies did not exhibit any synergy.
Modjtahedi et al (Cell Biophysics vol 22, 1993, 129-146) has tested combinations of several rat anti-EGFR antibodies with non-overlapping epitopes. The antibodies were of different isotypes. In all cases the effect of using two antibodies was intermediate between the effects of using similar amounts of the two monoclonal antibodies alone. This was confirmed both in vivo and in vitro.
WO 2004/032960 (Merck Patent) discloses that the combined use of two monoclonal antibodies, Mab425 and Mab225 (Cetuximab), results in an increased amount of antibodies bound to the surface of EGFR expressing cancer cells compared to a similar amount of each of the monoclonal antibodies alone. The publication also discloses increased down-regulation of EGFR when using the combination of antibodies compared to the two monoclonal antibodies.
Perera et al (Clin Cancer Res 2005; 11(17):6390-99) disclosed a synergistic effect of treating mice bearing U87MG.de2-7 xenografts with a combination of two murine monoclonal antibodies. One of the antibodies (mAb 528) binds all of the EGFR subtypes with similar specificity to cetuximab. The other one (mAB 806) only binds the de2-7 EGFR. The U87MG.de2-7 cell line is a de2-7EGFR transfected cell line. The U87MG.DK cell line expresses a kinase inactive variant of the de2-7 EGFR. No synergy was observed when the two antibodies were used against mice bearing U87MG.DK xenografts. In a xenograft model with the A431 cell line expressing wildtype EGFR, the authors provided no evidence of synergy. The de2-7 EGFR is only present in a limited number of cancer types, such as glioma, to some extent breast cancer and lung cancers.
While these studies have indicated that in some cases synergy may exist between two murine monoclonal antibodies, they also show that in many cases, no synergy is seen. The studies also do not provide an anti-EGFR antibody composition that is effective against a wide range of clinically relevant cancer cell lines.
Accordingly, the need exists for improved therapeutic antibodies against EGFR which are effective at treating and/or preventing diseases related to overexpression of EGFR when administered at low dosages. There is also a need for broadly applicable therapeutic cancer-antibodies which can be used without possessing intimate knowledge about the structure of EGFR expressed by the cancer cells in question.